Changes in levels of bio-chemicals and secondary metabolites during peak stress in field cassava

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Presented by Ephraim Nuwamanya, Patrick R Rubaihayo, Settumba Mukasa, Samuel Kyamanywa, Robert Kawuki, Joseph Hawumba and Yona Baguma (NaCRRI/MAK) at the First Bio-Innovate Regional Scientific Conference, Addis Ababa, Ethiopia, 25-27 February 2013

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Changes in levels of bio-chemicals and secondary metabolites during peak stress in field cassava

  1. 1. Changes in levels of bio-chemicals and secondary metabolites during peak stress in field cassavaEphraim Nuwamanya, Patrick R Rubaihayo, Settumba Mukasa, Samuel Kyamanywa, Robert Kawuki, Joseph Hawumba and Yona Baguma* NaCRRI/MAK First Bio-Innovate Regional Scientific Conference United Nations Conference Centre (UNCC-ECA) Addis Ababa, Ethiopia, 25-27 February 2013
  2. 2. Cassava with drought toleranceThe rationale for breeding cassava with drought tolerance is built in the fact that there is need to improve the inherent capacity of cassava as a food security and famine crop particularly in Sub-Saharan Africa, where millions of people suffer from chronic food shortages (Elsharkway, 1993)
  3. 3. Drought and temperature stress• Drought stress = – lack of precipitation over a period of time (Willwhite et al., 2007) or severe low soil moisture that denies plant access to water• Heat stress = increase in Temp above threshold for a period sufficient to cause damage to plant• Mitigation measures include avoidance or tolerance mechanisms
  4. 4. Wang et al., , Planta, 2003
  5. 5. Problem statement• In Uganda, no cassava varieties have been selected for tolerance to heat &moisture stress• No efforts have been put forward to understand mechanisms behind observed tolerance/resistance• This has hampered breeding efforts to improve cassava for tolerance to a number of abiotic stresses
  6. 6. Materials and experimental Layout• 20 Cassava varieties selected from different parts of country (www.selectionsfordrought tolerance study.ne)• Field experiment will be set up in Kasese Western Uganda (Gmap Kasese)• Lay out will be RCBD with 4 replications, 2 stressed and 2 control (irrigated) (exptal layout)•
  7. 7. Why Kasese?. Weather Characteristics In addition to receiving low rainfall, the trial site is in the rift valley experiencing higherthan average temperatures compared to the rest of Kasese district and the shallow soils allow for easy drainage of water increasing moisture stress
  8. 8. Soil properties at the trial siteProperty Units Content RangepH - 6.53 6.4-6.7Organic matter % 4.01 3.5-4.8Nitrogen % 0.22 0.2-0.25Phosphorus Ppm 23.80 6.0-55Calcium Ppm 4090.26 2316.4-7573Magnesium Ppm 882.3 346.3-1467.5Potassium Ppm 383.98 194.8-675.1Boron Ppm 1750 12.87-26.8Zinc Ppm 0.58 0.2-1.1Copper Ppm 10.23 8.2-13.8Manganese Ppm 156.34 82.0-235.7Iron Ppm 457.57 380.7-1786Sand % 53.47 49.8-59.8Clay % 21.90 24.9-30.9Silt % 18.63 15.3-21.3
  9. 9. Tissue and Organ Sampling Guide• Leaves• All measurements to be done on 5th fully expanded leaf unless otherwise – Leaf discs will be cut out of the 5th fully expanded leaf – Preservation in liquid nitrogen during transfer to the lab.• Stems and roots• All measurements to be whole plant parts sampled by taking samples on the distal, middle and posterior parts of the plant parts – Discs will be cut out of the root – Preservation in liquid nitrogen
  10. 10. Carbohydrates, Proteins and Pigments • Soluble sugars (hexoses) (Dubois et al., 1956; modified) • Starch content (Megazyme starch kit) • Cyanide content according to Onwuka – Cyanide (mg/g) = Absorbance x GF x DF Sample weight – Where: GF = gradient factor and DF = dilution factor• Concentrations of chlorophylls and carotenoids • determined in 100 % acetone (Wettshtein 1957) • at 662, 644 and 440.5 nm for chlorophyll a, chlorophyll b and carotenoids, respectively.
  11. 11. Establishment of peak stress• Described as the time between the onset of severe drought up to when relative humidity levels go up.• A period of less than 40% RH, less than 20% soil moisture and daily average temperatures above 340C.• A period between in 9MAP and 11MAP spanning over a five week period
  12. 12. A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4Plate 2: Physiological differences in the stay green (A), Susceptible (B) and early recovering (C) varieties pre-stress (1), 3 weeks post stress (2), 5 weeks post stress (3) and 7weeks post stress (4)
  13. 13. A B C Plate 1: Contrasting phenotypes dependent on physiologicalmechanism for tolerance to drought. A: Stay green, B: Susceptible and C: Early recovering varieties at 6-8 weeks post stress (peak stress)
  14. 14. Phenotypic observations during peak stress
  15. 15. Changes in starch content and FRS • Differences in rate of reduction or accumulation of starch and Reducing sugars were observedGroup H1SC H1FRS H2SC H2FRS H3SC H3FRS H4SC H4FRS RSC RFRS R2SC R2FRSAll Varieties 0.125 ± 0.007 0.151± 0.027 0.137± 0.02 0.217± 0.028 0.070± 0.029 0.179± 0.026 0.087± 0.013 0.206± 0.101 -0.017 0.013 0.6 0.308Stay Green 0.133± 0.005 0.134± 0.029 0.177± 0.039 0.240± 0.008 0.075± 0.015 0.182± 0.012 0.077± 0.005 0.231± 0.027 -0.023 0.023 0.74 0.377Early Recovering 0.106± 0.026 0.171± 0.033 0.111± 0.006 0.195± 0.004 0.071± 0.000 0.166± 0.023 0.075± 0.002 0.142± 0.001 -0.014 -0.012 0.64 0.48Susceptible 0.106± 0.016 0.168± 0.029 0.134± 0.002 0.227± 0.003 0.070 ± 0.013 0.170± 0.069 0.071± 0.010 0.239± 0.004 -0.017 0.018 0.502 0.29Table 1: H=Harvest 1, 2,3,and 4 corresponding to week 2, 4, 6 and 8 during stress. SC=Starch Content, FRS=Free Reducing Sugars, R=Rate of change in SC or FRS during the 8 weeks of stress.R2 = Regression Coefficient for changes in the different parameters during the stress period.• Rate of reduction in starch content high for SGV but low for ERV• Rate of increase in reducing sugars was high for SGV and SV but low (infact –ve gradient) for ERV-low amylase activity for ERV• No remobilisation from the root observed for ERV compared to SGV• Differences in mechanism of tolerance confirmed
  16. 16. Results: Free Reducing Sugars (RS) 0.5 A 0.9 B 0.8 0.4 0.7 y = -0.0718x + 0.6025 y = 0.02x + 0.357 0.6 0.3 0.5 R² = 0.1433 Reducing Sugars R² = 0.477 0.2 Reducing Sugars 0.4 0.3 0.1 Linear (Reducing 0.2 Linear Sugars) 0.1 (Reducing 0 0 Sugars) 0.8 0.5 y = -0.005x + 0.407 C y = 0.1104x + 0.2565 D R² = 0.016 0.7 0.45 0.6 R² = 0.9271 0.4 0.35 0.5 0.3 reducing 0.4 Reducing Sugars 0.25 Sugars 0.3 0.2 0.15 0.2 Linear (Reducing 0.1 Linear 0.1 Sugars) 0.05 (reducing 0 0 Sugars)A=Changes in Reducing Sugars among stay green varieties B=Changes in Reducing Sugars for Early recovering varietiesC=Changes in Reducing Sugars for susceptible variety. D=Changes in reducing Sugars for all the varieties
  17. 17. Changes in peel and fresh root cyanide contents • Although differences in rate of increase in cyanide were observed, peel cyanide content was the most important in making inferences on plants physiological behaviorVariety group H1CR H1CP H2CR H2CP H3CR H3CP H4CR H4CP H5CR H5CP RCR RCP R2CR R2CPAll varieties 0.332± 0.171 0.522± 0.339± 0.175 0.552±0.4 0.639± 1.269±0. 0.349± 0.557± 0.329 0.258±0.201 0.646± 0.613 -0.014 0.025 0.023 0.016 0.207 51 0.513 781 0.158Stay green varieties 0.393± 0.201 0.393± 0.309± 0.029 0.298± 0.549± 0.984± 0.347± 0.501± 0.344 0.356± 0.327 0.646± 0.571 -0.003 0.071 0.003 0.176 0.132 0.144 0.202 0.448 0.125Early recovering 0.301± 0.022 0.602± 0.245± 0.087 0.535± 0.642± 1.457± 0.246± 0.407± 0.196 0.087± 0.010 0.149± 0.014 -0.043 -0.103 0.108 0.110varieties 0.216 0.072 0.034 0.327 0.237Susceptible 0.168± 0.134 0.386± 0.381± 0.022 0.510± 0.405± 1.605± 0.301± 0.667± 0.137 0.070± 0.118 0.286± 0.087 -0.028 -0.004 0.093 0.002varieties 0.121 0.038 0.027 0.022 0.104Table 2: Changes in the cyanide content levels from onset of peak stress up to recovery. H=harvest, CP=Cyanide content for the peel; CR=Cyanide content for the root. R= rate of change;R2=Regression • While increments were observed for peel cyanide content in SGV, reductions were observed in ERV • Minor reductions were observed for fresh root cyanide content in SGV over the peak stress period while significant reductions were observed for ERV • No significant differences were observed for cyanide in SV
  18. 18. Changes in Cyanide Content 1.2 B 1.4 A 1.2 1 y= 0.0713x + 0.3497 y = 0.025x + 0.633 R² = 0.1758 0.8 1 R² = 0.016 CnP Peel CnP Peel 0.6 CnP Root 0.8 CnP Root Linear (CnP Peel) 0.6 0.4 Linear (CnP Peel) Linear (CnP Root) 0.4 Linear (CnP Root) y = -0.003x + 0.399 0.2 R² = 0.002 y = -0.013x + 0.423 0.2 R² = 0.023 0 0 MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9) MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9) 1.6 C 1.8 D 1.4 1.6 1.2 1.4 y = -0.004x + 0.703 1 1.2 R² = 0.000 y=- 0.1034x + 0.9402 CnP Peel 1 CnP Peel 0.8 R² = 0.1097 CnP Root Cnp Root 0.8 0.6 Linear (CnP Peel) Linear (CnP Peel) 0.6 Linear (CnP Root) y = -0.027x + 0.347 Linear (Cnp Root) 0.4 0.4 R² = 0.093 y = -0.042x + 0.432 R² = 0.108 0.2 0.2 0 0 MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9) MC(WK1) MC(WK3) MC(WK5) MC(WK7) MC(WK9)A=Cyanide changes for all varieties throughout the stress period, B=Cyanide Change for stay green varieties, C= Cyanide change for early recoveringvarieties, D=Cyanide change for susceptible variety
  19. 19. Total Pigments; Chla, Chlb, Cart 0.6 Pigments for stay greens 0.5 Total pigment content0.45 0.5 y = -0.020x + 0.464 y = -0.040x + 0.514 0.4 R² = 0.324 R² = 0.680 0.4 Chla0.35 Chla Chlb 0.3 y = 0.021x + 0.151 Chlb 0.3 cart y = 0.013x + 0.1760.25 R² = 0.732 cart R² = 0.547 Linear (Chla) 0.2 Linear (Chla) 0.2 Linear (Chlb)0.15 Linear (Chlb) y = 0.004x + 0.080 y = 0.005x + 0.079 Linear (cart) R² = 0.128 0.1 R² = 0.300 Linear (cart) 0.10.05 0 0 Harvest1 Harvest2 Harvest 3 Harvest 4 Harvest1 Harvest 2 Harvest 3 Harvest 4 0.45 Graph for early recovering genotypes 0.6 0.4 Pigments for susceptible y = 0.025x + 0.313 0.35 R² = 0.868 0.5 y = -0.014x + 0.446 R² = 0.045 0.3 Chla 0.4 Chla y = 0.045x + 0.072 0.25 Chlb R² = 0.977 Chlb cart 0.3 cart 0.2 y = 0.007x + 0.169 Linear (Chla) Linear (Chla) R² = 0.029 0.15 y = 0.015x + 0.042 Linear (Chlb) 0.2 Linear (Chlb) R² = 0.961 Linear (cart) y = 0.000x + 0.084 Linear (cart) 0.1 R² = 0.000 0.1 0.05 0 0 Harvest1 Harvest2 Harvest 3 Harvest 4 Harvest1 Harvest2 Harvest 3 Harvest 4
  20. 20. Bound Reducing Sugars/Total carbohydrate• There was an increase in total metabolisable carbohydrates• Storage/structural carbohydrates reduced with progressive stress• Among SGV and SV, high increments in metabolisable carbohydrates were coupled to high reductions in storage carbohydrates• Among ERV, reductions were observed in free metabolisable carbohydrates while slight reductions were observed for storage carbohydrates
  21. 21. Changes in carbohydrate Profiles 0.4 Total carbohydrate metabolite changes 0.45 Carbohydrate profiles for Stay green0.35 0.4 y = 0.039x + 0.174 y = 0.009x + 0.251 R² = 0.432 0.35 0.3 R² = 0.0150.25 0.3 Free RS y = 0.012x + 0.156 Free RS Bound RS R² = 0.308 0.25 0.2 Bound RS Starch Content 0.2 Linear (Free RS)0.15 Starch Content y = 0.023x + 0.138 y = -0.017x + 0.15 Linear (Bound RS) R² = 0.377 R² = 0.6 Linear (Free RS) 0.15 Linear (Starch Content) 0.1 Linear (Bound RS) y = -0.023x + 0.164 Linear (Starch Content) 0.10.05 R² = 0.743 Linear (Starch Content) 0.05 0 Harvest 1 Harvest 2 Harvest 3 Harvest 4 0 Harvest 1 Harvest 2 Harvest 3 Harvest 4 0.3 Carbohydrate profile for Early recovering 0.5 Carbohydrate profiles for susceptible varieties0.25 0.45 y = -0.006x + 0.216 R² = 0.067 0.4 0.2 y = -0.005x + 0.278 Free RS 0.35 R² = 0.002 Free RS Bound RS 0.3 Bound RS0.15 Starch Content y = -0.011x + 0.197 0.25 Starch Content R² = 0.474 Linear (Free RS) Linear (Free RS) 0.1 Linear (Bound RS) 0.2 y = -0.013x + 0.126 y = 0.015x + 0.162 Linear (Bound RS) R² = 0.634 Linear (Starch Content) 0.15 R² = 0.291 Linear (Starch Content)0.05 Linear (Starch Content) 0.1 y = -0.016x + 0.137 R² = 0.502 0.05 0 Harvest 1 Harvest 2 Harvest 3 Harvest 4 0 Harvest 1 Harvest 2 Harvest 3 Harvest 4
  22. 22. Conclusions• Peak stress is characterized by varied physiological responses involving modification in carbohydrate and Nitrogen metabolism• Varieties respond differently during peak stress showing varied mechanisms of tolerance to stress• Important parameters for consideration in understanding the levels of tolerance include total bound and free sugars, cyanide content as a secondary metabolite, and free metabolisable sugars
  23. 23. •Thank you•Asante sana

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